Vicilinas de sementes de leguminosas selvagens:purifica??o, caracteriza??o, efeito de delet?rio e mecanismo de a??o para bruqu?deos e para fungos fitopatog?nicos

• Main Author: Teixeira, Fabiano de Moura
• Other Authors: CPF:29706106391
• Format: Others
• Language: Portuguese
• Published: Universidade Federal do Rio Grande do Norte 2014
• Subjects: Sementes; Maculatus; Subfasciatus; Vicilinas; Seeds; Vacilin; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
• Online Access: http://repositorio.ufrn.br:8080/jspui/handle/123456789/12604

Made available in DSpace on 2014-12-17T14:03:41Z (GMT). No. of bitstreams: 1 FabianoMT.pdf: 637738 bytes, checksum: 985b4ebc65a8511464495683b08649e2 (MD5) Previous issue date: 2006-06-27 === Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior === Globulins fractions of legume seeds of Crotalaria pallida, Erytrina veluntina and Enterolobium contortisiliquum were isolated and submitted to assays against serine, cysteine and aspartic proteinases, as also amylase present in midgut of C. maculatus and Z. subfasciatus. Hemagglutination assays indicated presence of a lectin in E. veluntina globulin fractions. This lectin had affinity to human erythrocytes type A, B and O. Vicilins were purified by chromatography on Sephacryl S-300 followed of a chromatography on Sephacryl S-200, which was calibrated using protein markers. Vicilins from C. pallida (CpV) and E. veluntina (EvV) seeds had a molecular mass of 124.6 kDa and E. contortisiliquum a molecular mass of 151kDa. Eletrophoresis in presence of SDS showed that CpV was constituted by four subunities with apparent molecular mass of 66, 63, 57 and 45 kDa, EvV with three subunities with apparent molecular mass of 45kDa and EcV four subunities, two with 37.1 kDa and two with 25.8 kDa. Non denaturantig eletrophoresis displayed single bands with high homogeneity, where CpV had lower acidic behavior. All vicilins are glycoproteins with carbohydrate contents at 1 to1.5%. Bioassays were done to detect deleterious effects of vicilins against C. maculatus and Z. subfasciatus larvae. CpV, EvV and EcV exhibited a WD50 of 0.28, 0.19 and 1.03%; LD50 0.2, 0.26, and 1.11% respectively to C. maculatus. The dose responses of CpV, EvV and EcV to Z. subfasciatus were: WD50 of 0.12, 0.14, 0.65% and LD50 of 0.09, 0.1, and 0.43% respectively. The mechanism of action of these proteins to bruchids should be based on their properties of bind to chitin present in mid gut of larvae associated with the low digestibility of vicilin. In assays against phytopatogenous fungus, only EcV was capable of inhibit F. solani growth at concentrations of 10 and 20 ?g and its action mechanism should be also based in the affinity of EcV to chitin present in the fungi wall === Sementes de leguminosas das esp?cies Crotalaria pallida, Erythrina veluntina e Enterolobium contortisiliquum tiveram suas fra??es globul?nicas isoladas. As globulinas n?o apresentaram atividade de inibidores para as enzimas tipo ser?nicas, ciste?nicas, asp?rticas e para as amilases de homogenatos intestinais de larvas de Callosobruchus maculatus e Zabrotes subfasciatus. Os testes de atividades hemaglutinantes indicaram que apenas a globulina de Erytrina veluntina apresentou contamina??o com lectinas com afinidade para eritr?citos humanos do tipo A, B e O. As vicilinas foram ent?o purificadas usando-se duas cromatografias de filtra??o em gel, Sephacryl S-300 seguido de uma Sephacryl S-200 calibrada com marcadores de massa molecular conhecidos. Vicilinas de C. pallida (CpV) e E. veluntina (EvV) apresentaram massa molecular de 124,6 kDa e a de E. contortisiliquum (EcV) com massa molecular de 151 kDa..Eletroforese desnaturante mostrou que CpV possui 4 subunidades de massas moleculares aparentes de 66, 63, 57, 45 kDa; a de EvV com duas subunidades de massa molecular aparente de 66,2 e 63,8 kDa e EcV tr?s subunidades de massa molecular aparente de 54,9 kDa. Essas prote?nas mostraram ser homog?neas por eletroforese nativa com bandas ?nicas, onde CpV revelou ser a prote?na menos ?cida. Todas as vicilinas s?o glicoprote?nas,com conte?do de carboidrato variando de 1 a 1,5%. Bioensaios foram feitos para determinar o efeito delet?rio das vicilinas para C. maculatus e Z. subfasciatus. Cpv, EvV e EcV apresentaram WD50 de 0,28, 0,19 e 1,03%; LD50 de 0,2, 0,26 e 1,11% para C. maculatus, respectivamente . As curvas dose resposta para Cpv, EvV e EcV para Z. subfasciatus apresentou WD50 de 0,12, 0,14 e 0,65%; e LD50 de 0,09, 0,10 e 0,43%, respectivamente. Ensaios digest?rios utilizando se enzimas dos intestinos m?dio de bruqu?deos mostraram que as vicilinas s?o resistentes ? prote?lise, dentre elas destaca-se EcV, que mesmo depois de incubadas por 24 horas n?o foram hidrolisados pelas enzimas de de C. maculatus. Nos ensaios para fungos fitopat?genos apenas EcV foi capaz de inibir a germina??o de esporos de Fusarium solani ? concentra??o de 10 e 20 ?g. O mecanismo de a??o proposto para as vicilinas foi baseado na propriedade que essas prote?nas tiveram de se ligarem a quitina ou estruturas quitinosas presentes nos intestinos m?dios das larvas associado ? baixa digestibilidade destas prote?nas para enzimas dos insetos